Effective isolation of nucleic acids from biological samples (e.g., cultured cells, tissue, viruses) is an essential prerequisite for efficient downstream amplification, detection, and quantification of specific genetic sequences via quantitative polymerase chain reaction (qPCR). The extraction process requires lysing the cells with harsh extraction reagents, such as detergents or enzymes, thereby resulting in a mixture of nucleic acids, cellular debris and extraction reagents. The nucleic acids are then separated/purified from the cellular debris and extraction reagents using a variety of techniques (e.g. organic solvent extraction, chromatography, centrifugation, dialysis). These techniques can be very time-consuming, tedious, and often require multiple washing steps. By way of example, commercially-available nucleic acid isolation kits require approximately 15 minutes to over one hour to complete, largely due to the multiple washing steps required to sufficiently separate the nucleic acids from the cellular debris and extraction reagents. Consequently, it has been suggested that as much as 15% of all molecular biology research time is devoted to purification.
In view of the foregoing, various attempts have been made to reduce the time associated with isolating nucleic acids from a biological sample. By way of example, Kelso, United States Patent Application No. 20090246782 discloses a system, device, and method for performing biological reactions. More specifically, the system contemplates placing a sample in a first chamber. The first chamber includes first processing reagents to generate a processed sample. The processed sample is moved through a water and alcohol immiscible, hydrophobic, or lipophilic barrier to a second chamber. The processed sample is treated in said second chamber with second processing reagents to generate a further processed sample.
While functional for its intended purpose, the system disclosed in the '782 application has certain limitations. For example, the reagents and immiscible phase of the system disclosed in the '782 application must be confined within corresponding chambers. As a result, the system requires the use of an external pump or two-axis magnet to move the processed sample between the chambers. It can be appreciated that the use of an external pump may have undesired effects on the sample. Alternatively, the use of a two-axis magnet may add unwanted cost and complexity to the system. In addition, the use of a plurality of chambers to isolate the nucleic acids from a biological sample may limit the throughput of the system.
Shikida et al., United States Patent Application No. 20080226500 discloses a miniaturized chemical analytic apparatus. The apparatus includes an introduction means for introducing a first droplet having a specimen therein into a liquid such that the structure of the first droplet is maintained. Magnetic ultrafine particles are mixed into the droplet while, once again, maintaining the structure thereof. It is contemplated for the specimens in the first droplet to bind to the particles. Thereafter, a magnetic field conveys the first droplet, with the specimen-bound magnetic particles therein, through the liquid to a desired location wherein the first droplet may be united with a second droplet for further processing downstream. The process may be repeated. A bulkhead may be provided between the areas wherein the first and second droplets are formed to maintain the droplets in such area.
It is noted that while the apparatus disclosed in the '500 application contemplates the movement of specimen-bound magnetic particles through a liquid for processing, no mechanism is provided for extracting the specimen-bound magnetic particles from the first droplet, or the droplets in which the first droplet subsequently merges. Hence, the apparatus and methodology disclosed in the '500 patent does not provide for an effective means for isolating particles, and their associated analyte(s), from a biological sample.
Therefore, it is a primary object and feature of the present invention to provide a device for and a method of extracting and purifying a fraction from cultured cells, tissue samples and other biological materials.
It is a further object and feature of the present invention to provide a device for and a method of extracting and purifying a fraction from cultured cells, tissue samples and other biological materials that is simpler and more efficient than prior devices and methods.
It is a still further object and feature of the present invention to provide a device for and a method of extracting and purifying a fraction from cultured cells, tissue samples and other biological materials that has higher throughput than prior devices and methods.
In accordance with the present invention, a device is provided for facilitating extraction of a fraction from a biological sample. The biological sample includes non-desired material and a fraction-bound solid phase substrate. The device includes an input zone for receiving the biological sample therein and a phase-gate zone for receiving an isolation buffer therein. An output zone receives a reagent therein. A force is movable between a first position adjacent the input zone and a second position adjacent the output zone. The force urges the fraction-bound solid phase substrate from the input zone, through the phase-gate zone and into the output zone.